Systems and methods for generation of emulsions with suitable clarity with applications of use

ABSTRACT

The inventions cover systems and methods for generation of emulsions having suitable clarity without requiring refractive index matching between emulsion components. Systems can include: a substrate including a set of openings; a reservoir facing the substrate at a first side and containing a sample fluid configured for droplet formation upon interacting with the set of openings of the substrate; and a collecting container facing the substrate at a second side and containing a set of fluid layers configured with a density gradient and suitable immiscibility characteristics. One or more components of the system(s) can support methods for emulsion generation, in relation to enabling interactions between multiple continuous phases and a dispersed droplet phase to generate clear emulsions. Applications of the inventions(s) can include performance of droplet-based digital PCR in an improved manner (e.g., without requiring implementation of correction factors based upon Poisson statistics).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/561,608, filed Dec. 23, 2021, which is a continuation of U.S.application Ser. No. 17/230,907, filed Apr. 14, 2021, now U.S. Pat. No.11,242,558, issued Feb. 8, 2022, which claims the benefit of U.S.Provisional Application No. 63/010,490, filed Apr. 15, 2020, each ofwhich is incorporated in its entirety herein by this reference.

TECHNICAL FIELD

This invention relates generally to fields related to sample processing,and more specifically to a new and useful system and method forgeneration of an emulsion with suitable clarity in fields related tosample processing.

BACKGROUND

An emulsion is composed of droplets of a first fluid (i.e., dispersedphase) in a second fluid (i.e., continuous phase) that is immisciblewith the first fluid. Emulsions are typically cloudy due to scatteringof light at surfaces of the droplets. Clarity of emulsions can beimproved by aligning the refractive indices of the immiscible phases,thereby mitigating light scattering effects. Clarity of emulsions canalso be improved by forming microemulsions or nanoemulsions, in whichthe characteristic droplet size is less than the wavelength(s) of lightbeing used to irradiate the emulsion (e.g., less than 100 nm).

In biotechnology applications (e.g., cell-related applications, nucleicacid-derived analyses, protein analyses, etc.), however, useful dropletsizes are typically greater than 1 micron in diameter. In order togenerate emulsions with suitable clarity, one has to match therefractive indices of the emulsion fluid components (e.g., of thedispersed phase and the continuous phase). However, this approachrequires small tolerances in relation to matching of refractive indices.Furthermore, such an approach is extremely sensitive to user error. Forinstance, for compositions where the refractive indices are tuned byaddition of compounds, minor pipetting errors can cause the refractiveindex of the final mixture to fall outside of a suitable range.Refractive indices also change with temperature, and the degree ofchange can be different for different types of fluid. Thus, there arealso typically finite temperature ranges in which an emulsion remainsclear, which may not be appropriate for applications requiringmodifications to the temperature of the emulsion.

Thus, there is a need in the field of sample processing to create a newand useful system and method for generation of an emulsion havingsuitable clarity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts an embodiment of a system for generating an emulsion.

FIG. 1B depicts an alternative embodiment of a system for generating anemulsion.

FIG. 2 depicts a specific example of a system for generating anemulsion.

FIGS. 3A-3B depict variations of a system configuration for generatingan emulsion, where FIG. 3A depicts submersion of a droplet-generatingcomponent in a liquid layer, and FIG. 3B depicts separation of thedroplet-generating component from the liquid layer by a gas.

FIGS. 4A-4B depict variations of emulsions, where FIG. 4A depicts awater-in-oil-in-water emulsion and FIG. 4B depicts anoil-in-water-in-oil emulsion.

FIGS. 5A-5C depict variations of equilibrium emulsion positions within acollecting container.

FIG. 6A depicts a flowchart of a first embodiment of a method forgenerating an emulsion.

FIG. 6B depicts schematics of a variation of the first embodiment of amethod for generating an emulsion.

FIG. 6C depicts a flowchart of a second embodiment of a method forgenerating an emulsion.

FIGS. 6D-6E depicts schematics of variations of the second embodiment ofa method for generating an emulsion.

FIG. 7A depicts cross-sectional images of a collecting container (e.g.,cross-sectional images toward the center of the collecting container)containing an emulsion generated according to the method, where dropletsof the emulsion fluoresce in a manner that is detectable due to clarityof the emulsion.

FIG. 7B depicts an example of comparative performance of embodiments ofthe system/method described, in relation to conventional digital PCRplatforms that have high partitioning error due to limitations in thenumber of partitions provided.

FIG. 7C depicts a flow chart of an embodiment of a method for performingdroplet digital PCR.

FIG. 7D depicts a flow chart of a variation of a method for performingdroplet digital PCR.

FIG. 7E depicts a schematic of an embodiment of a quantificationoperation in accordance with the method shown in FIG. 7D.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. Benefits

The inventions associated with the system and method can confer severalbenefits over conventional systems and methods, and such inventions arefurther implemented into many practical applications across variousdisciplines.

The invention(s) can provide methods and systems for forming clearemulsions with droplet sizes greater than wavelengths of light used tointerrogate the emulsions for various applications, without requiringmatching of the refractive indices of emulsion components (e.g., adispersed phase and a continuous phase). Such invention(s) enable robustformation of emulsions having suitable clarity, with processes that areless subject to manual error (e.g., pipetting error). In particular, theinvention(s) create emulsions with droplets having continuous fluidlayers that are much thinner than the wavelength(s) of light being usedto irradiate them, whereby producing clear emulsions without requiringrefractive index matching for the various fluid/liquid phases used toproduce the droplets.

The invention(s) can also use centrifugation forces and/or other forcesto disperse a fluid, as droplets, through multiple layers of fluids(i.e., ‘continuous fluids’) that are immiscible with each other, formingat least a double emulsion. The dispersed fluid is immiscible with thefirst continuous fluid that it encounters but can be miscible with oneor more subsequent fluids it encounters. In embodiments, the dispersedfluid has a density configured to allow droplets to sink to a desiredposition within a collecting container. In embodiments, the refractiveindices of the various phases do not have to match, and the resultantemulsion is clear. This feature of the invention is attributed to thethickness of the continuous fluid surrounding each droplet being lessthan the characteristic wavelength(s) of light used to interrogate thesample.

The invention(s) also provide methods and systems for generating clearemulsions using multiple continuous fluid phases, each having differentdensity characteristics, where the clear emulsions are generated uponinteracting formed droplets with the multiple continuous fluid phases.

The invention(s) also provide methods and systems for forming clearemulsions with discrete droplets that are stable across ranges oftemperatures for various applications (e.g., nucleic acid processing andanalysis applications).

The invention(s) also provide methods and systems for positioningdispersed fluid droplets away from regions of fluid (e.g., regionscontaining surfactant micelles) that would otherwise prevent assessmentsby imaging-based interrogation modalities.

The invention(s) can also produce higher packing efficiencies thanclose-packing due to the thin layer of continuous phase about thedispersed phase. As such, higher packing efficiencies can enableapplications where a higher percentage of the dispersed phase relativeto the continuous phase is desired.

In particular, in embodiments, due to the high packing efficiency ofdroplets and the clarity of the emulsion in a container, theinvention(s) can produce and enable fast (e.g. <a few minutes) opticalanalysis of a high number of droplets (e.g., from 1 million to 100million droplets) per unit volume (e.g., 10 microliter through 100microliters), each droplet having a characteristic diameter (e.g., 10micron through 100 microns). In one example, the invention(s) canproduce and enable fast (e.g. <a few minutes) analysis of a high numberof droplets (e.g., 3.5 million droplets having a characteristic dropletdiameter of 30 micron within a 50 microliter volume). In anotherexample, the invention(s) can produce a high number of droplets (e.g.,˜28 million droplets having a characteristic droplet diameter of 15micron within a 50 microliter volume). However, variations can produceother numbers of droplets having other suitable characteristic diameterswithin other suitable collection volume sizes.

Applications of the invention(s) can include digital counting of nucleicacid molecules, peptide/protein molecules, cells, viral particles,living organisms, and/or other targets over a large dynamic range withlow error, due to the large number of droplets available forpartitioning and rapid analysis of a large number of droplets due to theclear emulsion being highly packed. In particular, the invention(s)allow droplet-based digital counting (e.g., digital PCR for analysis ofnucleic acid molecules) without requiring counting of negativecompartments (e.g., compartments not emitting a positive signalassociated with target material), and thereby without implementingcorrection factors (e.g., correction factors based on Poissonstatistics) for partitioning error, given that digital analysis hereincan be performed in a low occupancy (e.g., ˜5% or less) regime due tothe availability of large number of partitions. For instance, with ˜28million droplets in a 50 microliter reaction (mean diameter ˜15 micron),at ˜5% of occupancy, as many as ˜1.4 million nucleic acid molecules canbe analyzed in a digital PCR reaction enabled by the invention(s)without having to apply Poisson statistics to correct for partitioningerror.

Additionally or alternatively, the system and/or method can confer anyother suitable benefit.

2. System

As shown in FIG. 1A, an embodiment of a system 100 for generation of anemulsion includes: a substrate 110 including a set of openings 115; areservoir 120 facing the substrate at a first side and containing asample fluid 130 configured for droplet formation upon interacting withthe set of openings 115 of the substrate 110; and a collecting container140 facing the substrate 110 at a second side and containing a set offluid layers 150, wherein the set of fluid layers 150 is configured witha density gradient, wherein each fluid layer of the set of fluid layers150 is immiscible with adjacent fluid layers of the set of fluid layers,wherein at least one of the set of fluid layers is configured to providea thin film about individual droplets derived from the sample fluid,thereby producing clarity of the emulsion without refractive indexmatching of components of the emulsion, and wherein one or more of theset of fluid layers 150 is configured to provide a continuous phasesurrounding a disperse phase of droplets derived from the sample fluid130.

Alternatively, as shown in FIG. 1B, a related embodiment of a system 200for generation of an emulsion can include: a sample fluid 230 configuredfor droplet formation; and a collecting container 240 containing a setof fluid layers 250, wherein the set of fluid layers 250 is configuredwith a density gradient, wherein each fluid layer of the set of fluidlayers 250 is immiscible with adjacent fluid layers of the set of fluidlayers 250, wherein at least one of the set of fluid layers isconfigured to provide a thin film about individual droplets derived fromthe sample fluid, thereby producing clarity of the emulsion withoutrefractive index matching of components of the emulsion, and wherein oneor more of the set of fluid layers 250 is configured to provide acontinuous phase surrounding a disperse phase of droplets derived fromthe sample fluid 230.

Embodiments of the system 100 function to form emulsions having suitableclarity for interrogation (e.g., by an imaging apparatus) withoutrequiring refractive index (RI) matching between fluid components of theemulsions. Embodiments of the system 100 also function to facilitateprocesses that are less subject to manual error (e.g., pipetting error).Embodiments of the system 100 also function to form emulsions havingsuitable clarity and with discrete droplets that are stable acrossranges of temperatures for various applications (e.g., nucleic acidprocessing and analysis applications). The system 100 can also functionto position dispersed fluid droplets away from regions of fluid (e.g.,regions containing surfactant micelles) that would otherwise obstructinterrogation (e.g., by imaging apparatus).

In various applications, the system 100 can produce emulsions havingsuitable clarity for enabling readout of analytes encapsulated in thedispersed phase of the emulsion, within a closed container used tocollect the dispersed phase. Such a configuration mitigates sample lossand/or sample contamination that occurs using other methods ofgenerating emulsions. In variations, readout of fluorescent signals(e.g., from labeled analytes within droplets of the dispersed phase,from products of analytes within droplets of the dispersed phase, etc.)can be performed by one or more of a 3D scanning technique (e.g., lightsheet imaging, confocal microscopy, etc.) and a planar imaging technique(e.g., to take images of a cross-section of the closed container).Additionally or alternatively, in a some applications, readout ofcolorimetric changes associated with droplets of the dispersed phase canbe performed by 3D imaging techniques (e.g., 3D brightfield constructionusing light field imaging, etc.).

In other variations, readout of non-fluorescent signals from droplets ofthe dispersed phase can be performed. For instance, products resultingfrom reactions within individual droplets of the dispersed phase canproduce changes in refractive indices, light absorption, lightscattering, light reflection, light transmission, and/or other lightinteraction characteristics that are different from empty or unreacteddroplets, for detection by various techniques (e.g., spectrophotometrictechniques, turbidimetric techniques, etc.).

For instance, in relation to applications involving DNAs, RNAs, aptamersagainst proteins, chemical groups, and/or other targets, any one or moreof: labeling of a target analyte or amplified version of the targetanalyte with fluorescent probes or dyes (e.g., hydrolysis probes, TaqManprobes, fluorescence resonant energy transfer (FRET) probes, fluorescentin situ hybridization (FISH) probes, DNA intercalating dyes etc.),labeling of probes (e.g., using digoxigenin (DIG), using biotin),labeling by way of fluorescently labeled nucleotides, labeling withDNA/RNA/ssDNA-specific dyes, or detecting by-products of DNAamplification (e.g., pH changes using pH-sensitive dyes, pyrophosphatasebuild up, etc.) can produce detectable/amplifiable signals (e.g.,fluorescence, changes in color, with conjugation of an antibody ofalkaline phosphatase or peroxidase) that are detectable from droplets ofa dispersed phase produced according to the systems and methodsdescribed.

In relation to applications involving proteins, fluorescently labeledantibodies against target analytes or labeled probes (e.g., using DIG,using biotin) can produce detectable/amplifiable signals (e.g.,fluorescence, changes in color, with conjugation of an antibody ofalkaline phosphatase or peroxidase) that are detectable from droplets ofa dispersed phase produced according to the systems and methodsdescribed.

In relation to applications involving cells, organelles,micro-organisms, viruses, or exosomes, processing with stains (e.g.,live-dead stains), by cell type, by function, by detection of specificproteins (as described above), by DNAzymes, or by other processes canproduce detectable/amplifiable signals that are detectable from dropletsof a dispersed phase produced according to the systems and methodsdescribed.

In specific applications, the emulsion(s) generated by the systems andmethods described can be used for one or more of: detection and countingof nucleic acid molecules via amplification of individual nucleic acidmolecule captured within a droplet followed by detection of opticallydetectable signals (e.g., amplification by polymerase chain reaction(PCR) methods, by isothermal methods such as loop-mediated isothermalamplification (LAMP), by recombinase polymerase amplification (RPA), byhelicase dependent amplification (HDA), by strand displacementamplification (SDA), by nicking enzyme amplification (NEAR), bytranscription mediated amplification (TMA), by RNaseH mediatedamplification, by whole genome amplification (WGA) using phi29, byrolling circle amplification, etc.) on purified DNA, cDNA, RNA,oligonucleotide tagged antibodies/proteins/small molecules, or directlyfrom lysate (e.g., blood lysate); fluorescent in situ hybridization(FISH) with fluorescently tagged nucleic acids (e.g., PNA, LNA, DNA,RNA, etc.) or an indirect in situ hybridization approach using DIG orbiotin, where the signal is later amplified by conjugation of anantibody to alkaline phosphatase or a peroxidase to produce a change incolor detected by one or more substrates (e.g., nitroblue tetrazolium(NBT), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), HNPP, etc.); an invitro transcription or translation assay whereby a colorimetric orfluorescent reporter is used for detection; droplet PCR applied tosamples derived from single cells (e.g., prokaryotes, eukaryotes),organelles, viral particles, and exosomes; enumeration of protein orpeptide molecules (e.g., by proximity ligation assays, etc.); sequencingapplications (e.g., single molecule sequencing applications); monitoringor detection of products (e.g., proteins, chemicals) released fromsingle cells (e.g., interleukin released from immune cells); monitoringcell survival and/or division for single cells; monitoring or detectionof enzymatic reactions involving single cells; antibiotic resistancescreening for single bacteria; enumeration of pathogens in a sample(e.g., in relation to infections, sepsis, in relation to environmentaland food samples, etc.); enumeration of heterogeneous cell populationsin a sample; enumeration of individual cells or viral particles (e.g.,by encapsulating cells in droplets with species-specific antibodiescoupled with enzymes that react with substrate components in the dropletto produce signals, etc.); monitoring of viral infections of a singlehost cell; liquid biopsies and companion diagnostics; prenatal diagnosisof genetic disorders (e.g., aneuploidy, genetically inherited diseases)such as with cell-free nucleic acids, fetal cells, or samples containingmixtures of fetal and maternal cells; detection of cancer forms fromvarious biological samples (e.g., detection of cancer from cell-freenucleic acids, tissue biopsies, biological fluids, faeces); detectionand/or monitoring of minimal residual diseases; monitoring responses totherapies; detection or prediction of rejection events of transplantedorgans; other diagnostics associated with other health conditions; othercharacterizations of statuses of other organisms; and other suitableapplications.

In particular, in embodiments, due to the high packing efficiency ofdroplets and the clarity of the emulsion in a container, theinvention(s) can produce and enable fast (e.g. <a few minutes) opticalanalysis of a high number of droplets (e.g., from 1 million to 100million droplets) per unit volume (e.g., 10 microliter through 100microliters), each droplet having a characteristic diameter (e.g., 10micron through 100 microns). In one example, the invention(s) canproduce and enable fast (e.g. <a few minutes) analysis of a high numberof droplets (e.g., 3.5 million droplets having a characteristic dropletdiameter of 30 micron within a 50 microliter volume). In anotherexample, the invention(s) can produce a high number of droplets (e.g.,˜28 million droplets having a characteristic droplet diameter of 15micron within a 50 microliter volume). However, variations can produceother numbers of droplets having other suitable characteristic diameterswithin other suitable collection volume sizes.

Applications of the invention(s) can include digital counting of nucleicacid molecules, peptide/protein molecules, cells, viral particles,living organisms, and/or other targets over a large dynamic range withlow error, due to the large number of droplets available forpartitioning and rapid analysis of a large number of droplets due to theclear emulsion being highly packed. In particular, the invention(s)allow droplet-based digital counting (e.g., digital PCR for analysis ofnucleic acid molecules) without requiring counting of negativecompartments (e.g., compartments not emitting a positive signalassociated with target material), and thereby without implementingcorrection factors (e.g., correction factors based on Poissonstatistics) for partitioning error, given that digital analysis hereincan be performed in a low occupancy (e.g., ˜5% or less) regime due tothe availability of large number of partitions. For instance, with ˜28million droplets in a 50 microliter reaction (mean diameter ˜15 micron),at ˜5% of occupancy, as many as ˜1.4 million nucleic acid molecules canbe analyzed in a digital PCR reaction enabled by the invention(s)without having to apply Poisson statistics to correct for partitioningerror.

Extensions of the systems and methods described for producing emulsionswith suitable clarity can further be applied to other industries (e.g.,the cosmetic industry, food industries, other chemical processingindustries, etc.). Extensions of the systems and methods described canalso be applied to production of microparticles, where themicroparticles either have solid cores with functionalized surfaces(e.g. by surrounding droplets with a dispersed phase that can bepolymerized with functional groups in the first encountered fluid of theset of fluid layers) or hollow cores with shells (e.g., by surroundingdroplets of the dispersed phase with one or more layers carryingpolymerizable components, thereby forming a polymer shell about a fluidcore). Extensions of the systems and methods described can also beapplied to synthetic biology for generation of synthetic biologicalcomponents. For instance, the systems and methods can be adapted toproduce synthetic cells, giant unilamellar phospholipid vesicles (GUVs),polymersomes, liposomes, exosomes, multiple layer of synthetic cellsforming artificial tissue/organs, or other synthetic biology components.Extensions of the systems and methods can also be used to generatedispersions of microparticles captured within shells. Extensions of thesystems and methods described can also be applied to the pharmaceuticalindustry. For instance, the systems and methods described can generatecontrolled release drug solutions (e.g., by surrounding droplets of thedispersed phase that carry drug components with one or more layerscarrying reactants that can be used to trigger drug release by exposureto light, heat, changes in pH, chemical activators, mechanical forces,etc.). Additionally or alternatively, the systems and methods describedcan generate compositions where undesired tastes or odors of a corefluid are masked by a shell layer of fluid of the emulsion (e.g., withrespect to food and pharmaceutical applications). Additionally oralternatively, the systems and methods described can involve continuousphase-carrying reactants, where the reactants can diffuse into dropletsof the disperse phase when the droplets reach the continuous phase,thereby producing chemical reactions within the droplets of the dispersephase.

Extensions of the systems and methods described can produce higherpacking efficiencies than close-packing due to the thin layer ofcontinuous phase about the dispersed phase. As such, higher packingefficiencies can enable applications where a higher percentage of thedispersed phase relative to the continuous phase is desired.

Extensions of the systems and methods described can, however, be appliedto other fields as appropriate.

Embodiments, variations, and examples of the system 100 are configuredfor execution of one or more steps of the method 300 described inSection 3 below; however, embodiments, variations, and examples of thesystem 100 can alternatively be configured to execute another suitablemethod.

2.1 System—Substrate

As shown in FIG. 1A, the system 100 can include a substrate 110including a set of openings 115, which collectively function to producedroplets from a sample fluid when the sample fluid passes through theset of openings 115 (e.g., under applied force). As shown in FIG. 1A,the substrate 110 can be configured to interface with a reservoir 120containing the sample fluid 130, and to allow droplets generated by theset of openings 115 to be transmitted into the collecting container 140(described in more detail below).

The system 100 can be configured such that the sample fluid is driventhrough the opening(s) of the substrate 110 under an applied force. Invariations, the applied force can be attributed to one or more of anapplied pressure (e.g., pressurized gas driven by a pumping element,driven by a syringe, etc.), centrifugation, to applied electric fields,and other mechanisms for actively driving fluid flow. In specificexamples, the applied force can be applied by centrifugation apparatus,where centrifugation can be performed using a swing bucket (i.e., inwhich centrifugal forces are along the longitudinal axis of thecollecting container), or by using a fixed angle rotor (i.e., in whichcentrifugal forces are at an angle relative to the longitudinal axis ofthe collecting container). Additionally or alternatively, the system 100can be configured such that the sample fluid is flows through theopening(s) of the substrate 110 passively (e.g., due to normalgravitational forces, due to capillary effects, due to electric forces,etc.).

In variations, the substrate 110 can be configured as a plate, membrane,or mesh, and the set of openings 115 can include one or more openings,each having a characteristic dimension (e.g., width, diameter), thatranges from 0.05 um to 500 um. The openings can be uniform in morphologywith respect to each other, or can alternatively have non-uniformmorphology (e.g., with a defined average dimension having variabilitywithin a threshold tolerance level). Each of the one or more openingscan have a channel axis preferentially aligned with a longitudinal axisof the collecting container 140. Alternatively, at least one of the oneor more openings can have a channel axis that is non-parallel to (e.g.,at an angle to, transverse to, etc.) a longitudinal axis of thecollecting container 140, for instance, in applications involving stepemulsification. In variations, an opening of the one or more openingscan have a cross-sectional morphology that is constant along itsrespective channel axis; alternatively, an opening of the one or moreopenings can have a cross-sectional morphology that is non-constantalong its respective channel axis.

The substrate 110 can be retained in position by way of at least one ofthe reservoir 120 and the collecting container 140, or retained inanother suitable manner or by another suitable element of the system100.

The substrate 110 can be composed of a material having suitablemechanical properties (e.g., in terms of compliance, in terms ofhardness, in terms of moduli, etc.), surface properties (e.g.,hydrophobicity, hydrophilicity, porosity, charge, etc.), physicalproperties (e.g., in relation to reactivity with components of thesample fluid), in terms of optical properties, in terms of thermalproperties (e.g., in terms of heat transfer coefficients, in terms ofcoefficients of expansion, etc.), and/or other properties.

In variations, the substrate 110 can be composed of one or more of:glass, quartz, a metallic material (e.g., stainless steel), a polymericmaterial, or a natural material. In specific examples, the substrate 110can be composed of one or more of: glass (e.g., controlled pore glass,glass fiber, another silica-derived material), another porous glassmembrane (e.g., Shirasu porous glass membrane), a metal (e.g., aluminum,silver, stainless steel, etc.), a semiconductor-derived material (e.g.,silicon, silicon nitride), and a polymer (e.g., polyacrylonitrile, PVDF,cellulose acetate, polyester PETE, polyimide, PTFE, polycarbonate PCTE,polypropylene, etc.).

In embodiments, variations, and examples, the substrate 110 can includebe configured as described in U.S. application Ser. No. 16/309,092titled “Method for preparing micro-channel array plate, device forobtaining liquid drops using the micro-channel array plate, and methodfor generating liquid drops”, which is herein incorporated in itsentirety by this reference. Alternatively, the substrate 110 can beconfigured in another suitable manner.

Additionally or alternatively, in relation to FIG. 1B and other methodsdroplet generation, the system 100 can omit embodiments, variations, andexamples of the substrate 110 described above. For instance, the system100 can be configured to generate droplets from a sample fluid by way ofother microfluidic elements (e.g., channels), by use of needles (e.g.,stainless steel needle instruments, needles composed of anothersuitable/non-reactive material, etc., by use of capillary instrument(e.g., pulled glass capillary pipettes, other capillary instruments,etc.), by atomization, by electrodynamic droplet formation, or byanother suitable method, where the droplets are then used to form theemulsion(s) described.

2.2 System—Sample Fluid and Disperse Phase

As shown in FIG. 1A, the system 100 can include a reservoir 120 facingthe substrate 110 at a first side and containing a sample fluid 130configured for droplet formation upon interacting with the set ofopenings 115 of the substrate 110. The reservoir 120 functions tocontain the sample fluid prior to generation of droplets from the samplefluid (i.e., as a dispersed phase). The reservoir 120 can also functionto support the substrate 110, in order to provide an assembly thatinterfaces with the collecting tube 140 described below. In onevariation, as shown in FIG. 1A and FIG. 2 , the reservoir 120, 120′ caninclude a base portion that supports the substrate 110, 110′, and allowsthe sample fluid 130, 130′ to pass from the reservoir 120, 120′, throughthe set of openings of the substrate 110, 110′, and into the collectingcontainer 140, 140′. However, other variations of the reservoir 120 canbe configured to interface with the substrate 110 and/or the collectingcontainer 140 in another suitable manner.

In material composition, the reservoir 120 can be composed of one ormore of: a polymer (e.g., polypropylene, polydimethylsiloxane,polystyrene, polyvinyl chloride, polymethyl methacrylate, PEEK, ABSetc.), a silicon-derived material, glass, a metallic material, a ceramicmaterial, a natural material, a synthetic material, and/or any suitablematerial. The reservoir 120 can be composed of a material havingsuitable mechanical properties (e.g., in terms of compliance, in termsof hardness, in terms of moduli, etc.), surface properties (e.g.,hydrophobicity, hydrophilicity, porosity, charge, etc.), physicalproperties (e.g., in relation to reactivity with components of thesample fluid), in terms of optical properties, in terms of thermalproperties (e.g., in terms of heat transfer coefficients, in terms ofcoefficients of expansion, etc.), and/or other properties

In variations where the reservoir 120 is at least partially seatedwithin the collecting container 140, the reservoir 120 can include aprotrusion defining a set position of the reservoir 120 relative to anopening of the collecting container 140 (described in more detailbelow), such that the reservoir 120 does not move into or out of thecollecting container 140 in an undesired manner during operation. Insuch variations, the reservoir 120 can be configured to, with or withoutintermediate sealing elements, prevent fluid within the collectingcontainer 140 from leaving the collecting container 140 at the interfacebetween the reservoir 120 and the collecting container 140.

As described above, the reservoir 120 functions to contain the samplefluid prior to droplet generation as a dispersed phase of an emulsion.The sample fluid 130 can be an aqueous solution or can alternatively bea non-aqueous solution that is or is not miscible with water (e.g., oil,etc.), depending upon applications of use for the emulsion(s) generated.In first variations, the sample fluid 130 can be an aqueous solution. Inspecific examples of the first variations, the sample fluid 130 caninclude material associated with biological sample processing, such as:a mixture for PCR, a cell suspension, a suspension of organisms (e.g.,bacteria, viruses), a nucleic acid solution (e.g., a solution of DNA forgenomic amplification, a solution of RNA for reverse transcription,etc.), an amino acid or protein solution (e.g., a mixture of amino acidsfor synthesis, a mixture for protein crystallization, etc.), a mixturefor a gelation reaction, a reagent (e.g., buffer, lysing reagent,enzyme, etc.), or another suitable aqueous solution (e.g., for anotherapplication of use).

In second variations, the sample fluid 130 is a non-aqueous liquid, andin specific examples can be a silicone oil (e.g., oligomericdimethylsiloxane, cyclopentasiloxane, aliphatic siloxane, phenylsiloxane, fluorosiloxane, etc.), mineral oil, a hydrocarbon oil, afluorocarbon oil, a fluorosilicone oil, another oil, a fatty acidglyceride (e.g., glyceryl dilaurate, glyceryl oleate, glyceryllinoleate, glyceryl stearate, glyceryl isostearate, glyceryl sorbate,etc.), a non-aqueous solution associated with cosmetics production, anon-aqueous solution associated with food production, a non-aqueoussolution associated with a pharmaceutical application (e.g., a topicalmedication), or any other suitable non-aqueous liquid.

The sample fluid 130 can further be configured such that the density ofthe dispersed fluid is tuned in relation to the densities of one or moreof the fluids in the fluid layers 150 described in more detail below. Assuch, the density of the dispersed fluid can be tuned such that formeddroplets acquire a shell upon interaction with one or more of the fluidlayers 150 and sink to an equilibrium position relative to one or moreof the fluid layers 150 (e.g., after application of force bypressurization, by centrifugation, etc.). In one example, for awater-in-oil-in-water emulsion, the dispersed fluid can be tuned to havea density of >1, an oil layer can have a density <1, and an aqueouslayer can have a density of ˜1. In this example, the formed dropletswill sink to the bottom of the collecting container 140 after appliedforce and acquire a thin oil shell. The densities of aqueous solutionscan be tuned by adding different amounts of density medium (e.g.,Optiprep™, Histodenz™, Nycodenz™, heavy water or other suitable densityincreasing medium).

Additionally or alternatively, in other variations, the sample fluid 130can include a sample fluid as described in U.S. application Ser. No.16/309,093 titled “Oil-phase composition for generating water-in-oilliquid drops by means of centrifugation”, which is herein incorporatedin its entirety by this reference

However, in still other variations, the sample fluid 130 can include anyother suitable components and/or be composed of other materials.

2.3 System—Fluid Layers as Continuous Phase(s)

As shown in FIG. 1A, the system 100 includes a collecting container 140facing the substrate 110 at a second side and containing a set of fluidlayers 150, where the collecting container 140 functions to receivedroplets of the sample fluid 130, and to transmit the droplets intoand/or through one or more of the set of fluid layers 150 to generate atleast a double emulsion. The first fluid (other than gas) of the set offluid layers 150 that the dispersed fluid encounters is preferablyconfigured to be immiscible with the dispersed fluid. Furthermore, theset of fluid layers 150 functions to provide at least one stable thinfilm layer about each droplet of a disperse phase generated from thesample fluid 130 before the disperse phase arrives at its equilibriumposition within a continuous phase of the set of fluid layers 150. Assuch, the arrangement and composition(s) of the set of fluid layers 150functions to form at least double emulsions having suitable clarity fordownstream analyses, due to the thickness of the thin film layerssurrounding droplets being less than the wavelength(s) of light used toobserve the emulsions. As such, clarity of the resulting emulsions isnot attributed to refractive index (RI) matching between fluids used.The equilibrium position(s) of the droplets of the disperse phase at oneor more of the set of fluid layers 150 also allows interrogation of thedroplets (e.g., by using imaging apparatus, by other optical-basedinterrogation) for downstream applications and assessments, and allowsseparation of the emulsion from layers (e.g., layers containingsurfactant micelles) that prevent clear observation of the emulsion atits equilibrium position.

In variations, the collecting container 140 can be a containerconfigured for centrifugation (e.g., a centrifuge tube, amicrocentrifuge tube, etc.), a process container for PCR (e.g., a PCRtube), a strip tube, a plate having wells (e.g., a microtiter plate, amulti-well plate), or another suitable collecting container for theemulsion(s) generated. Alternatively, the collecting container 140 canbe a custom-designed container for generating and/or containing theemulsion prior to downstream analyses or further processing. Forinstance, the collecting container 140 can include one or more openings(e.g., with or without valves) for transmitting contents from thecollecting container 140 and/or for receiving additional material intothe collecting container 140. For instance, after an initial reaction orgeneration of a first emulsion, the system 100 can be configured toreceive additional materials (e.g., liquids, gases, etc.) to provideadditional reactions or processing steps.

In variations, the set of fluid layers 150 is configured with a densitygradient, and in the orientation shown in FIGS. 1A and 2 , the densestfluid of the set of fluid layers 150 is located at the base of thecollecting container 140, and the least dense fluid of the set of fluidlayers 150 is located furthest from the base of the collecting container140. As shown in FIG. 3A, the least dense layer of the set of fluidlayers 150 can be a liquid phase material, such that droplets of thesample fluid 130′ are transmitted directly into a first liquid layer151′ of the set of fluid layers without passing through another layer.In this variation, the substrate 110′ and/or reservoir 120′ describedabove can be at least partially submerged in the first liquid layer 151of the set of fluid layers 150. Alternatively, as shown in FIG. 3B, theleast dense layer of the set of fluid layers 150 can be a gas phasematerial (e.g., air, another gas), such that droplets of the samplefluid 130′ are transmitted into a first liquid layer 152 of the set offluid layers 150 by first passing through a gas layer. In thisvariation, the substrate 110 and/or reservoir 120 described above maynot be at least partially submerged in the first liquid layer 152 of theset of fluid layers 150 and may instead be separated from the firstliquid layer 152 by a gap (e.g., of air).

Furthermore, each fluid layer of the set of fluid layers 150 ispreferably immiscible with adjacent fluid layers of the set of fluidlayers 150, such that the layers of the set of fluid layers 150 aresubstantially discrete in relation to each other. Alternatively at leastone of the set of fluid layers 150 can be miscible with an adjacentfluid layer (e.g., but with a different density compared to the adjacentfluid layer), such that there is a non-discrete boundary between theadjacent fluid layers. Alternatively, the set of fluid layers caninclude adjacent fluid layers that are both aqueous solutions, butnon-miscible with each other (e.g., as aqueous multi-phase systems).

In relation to composition, alternating fluid layers of the set of fluidlayers 150 can include aqueous solutions and/or non-aqueous solutions.In variations, aqueous solutions of the set of fluid layers 150 caninclude material associated with biological sample processing, such as:material for PCR, a suspension of particles (e.g., particles withbinding moieties) or other biological material, a nucleic acid solution(e.g., a solution of DNA for genomic amplification, a solution of RNAfor reverse transcription, etc.), an amino acid or protein solution(e.g., a mixture of amino acids for synthesis, a mixture for proteincrystallization, etc.), a mixture for a gelation reaction, a reagent(e.g., buffer, lysing reagent, enzyme, etc.), or another suitableaqueous solution (e.g., for another application of use). In one example,an aqueous continuous phase can be composed of water/heavy water or asuitable buffer (e.g., saline buffer), where the dispersed phase (e.g.,droplets) are composed of a PCR mix with a density medium. Furthermore,in this example, a solute (e.g., simple sugar molecules) were added tothe aqueous phase in order to balance osmolarity of the aqueous solutionwith the PCR mix, in order to prevent osmosis or droplet expansion overtime.

In variations, non-aqueous liquids of the set of fluid layers 150 caninclude one or more of (e.g., including blends of): a silicone oil(e.g., oligomeric dimethylsiloxane, cyclopentasiloxane, aliphaticsiloxane, phenyl siloxane, fluorosiloxane, etc.), mineral oil, ahydrocarbon oil, a fluorocarbon oil, a fluorosilicone oil, another oil,a fatty acid glyceride (e.g., glyceryl dilaurate, glyceryl oleate,glyceryl linoleate, glyceryl stearate, glyceryl isostearate, glycerylsorbate, etc.), a non-aqueous solution associated with cosmeticsproduction, a non-aqueous solution associated with food production, anon-aqueous solution associated with a pharmaceutical application (e.g.,a topical medication), or any other suitable non-aqueous liquid.Additionally or alternatively, in variations, one or more of the set oflayers 150 can include a gas-phase material, a solid-phase material, orany other suitable phase of material.

In relation to generation of emulsions, one or more of the set of fluidlayers 150 (e.g., continuous phase(s) of the set of fluid layers 150)can include a surfactant, fluorosurfactant (e.g., containing aperfluoroalkyl group, etc.), and/or emulsifier to promote generation ofstable emulsions. In examples, the surfactant(s) and/or emulsifiers usedcan include one or more of: a Tween® (e.g., Tween® 20, Tween® 21, Tween®40, Tween® 60, Tween® 61, Tween® 65, Tween® 80, etc.), a Span® (e.g.,Span® 20, Span® 40, Span® 60, Span® 80, Span® 83, Span® 85, Span® 120,etc.), an Abil® (e.g., Abil® we09, Abil® em90, Abil® em120, Abil® em180,etc.), Dow Corning® 5200, Dow Corning ES-5612, Dow Corning® ES-5300, DowCorning ES-5600, Dow Corning® emulsifier 10, DehymuLs® SML, Cremophor®WO 7, Isolan® GI 34, Isolan® GIPDI, Tegosofit® Alkanol S 2, Triton™(e.g. Triton X-100), IGEPAL™ CA-630/NP-40, and another suitablesurfactant/fluorosurfactant/emulsifier. In one example, a non-aqueouscontinuous phase (e.g., non-aqueous continuous phase where there is adistribution of continuous phases used) can be composed of a siliconeoil blend with surfactants.

Additionally or alternatively, in other variations, one or more of theset of fluid layers can include a composition as described in U.S.application Ser. No. 16/309,093 titled “Oil-phase composition forgenerating water-in-oil liquid drops by means of centrifugation”, whichis herein incorporated in its entirety by this reference

In variations, the set of fluid layers 150 can include two fluid layers,three fluid layers, four fluid layers, or greater than four fluidlayers, where each layer has suitable thickness and/or volume to providesuitable amounts of transit time for droplets and/or amounts of materialfor generating stable emulsions. Additionally or alternatively, multiplefluid layers can be used to position generated droplets (e.g., generateddroplets and a bottom or top surface of the collecting container 140,140′, where methods described further below cover variations of methodsfor generating clear emulsions using multiple immiscible continuousphases having different densities in relation to density of thedispersed phase. In a first variation, as shown in FIG. 4A, the samplefluid is an aqueous solution (e.g., PCR mixture/solution) and the set offluid layers includes a first oil (to generate a thin film aboutdroplets of the sample fluid) and a second aqueous solution (e.g.,equilibrium continuous phase), which functions to receive droplets thathave passed through the first oil in order to form awater-in-oil-in-water (W/O/W) emulsion with suitable clarity. In anothervariation, as shown in FIG. 4B, the sample fluid 130 is a non-aqueoussolution that is immiscible with water, and the set of fluid layers 150includes a first aqueous solution (to generate a thin film aboutdroplets of the sample fluid) and a second non-aqueous solution (e.g.,equilibrium continuous phase of oil), which functions to receivedroplets that have passed through the first aqueous solution in order toform an oil-in-water-in-oil (O/W/O) emulsion with suitable clarity.However, in other still other variations, the set of fluid layers 150can include other suitable numbers and compositions of fluids forforming W/O/W/ . . . /W emulsions, W/O/W/ . . . /O emulsions, O/W/O/ . .. /O emulsions, or O/W/O/ . . . /W emulsions having any other suitablenumbers of layers of the disperse phase and an appropriate compositionof the continuous phase. Stable density gradients in the fluids used, aswell as having an oil film at the outermost layer of each droplet of thedispersed phase can contribute to emulsion stability as well.

As shown in FIG. 5A, the resulting density of the disperse phase afterinteracting with one or more fluid layers of the set of fluid layers canbe greater than densest layer of the set of fluid layers, such that theemulsion ultimately rests at the base of the collecting container.Alternatively, the resulting density of the disperse phase afterinteracting with one or more fluid layers of the set of fluid layers canbe less than that of the densest layer of the set of fluid layers, suchthat the emulsion does not ultimately rest at the base of the collectingcontainer. Still alternatively, as shown in FIG. 5C, in variations whereemulsions of different compositions are collected within a singlecollecting container, the arrangement and compositions of the set offluid layers can be configured such that the system 100 generates afirst emulsion that has an equilibrium position in one layer of the setof fluid layers, and a second emulsion that has an equilibrium positionin a second layer of the set of fluid layers.

The system 100 can, however, additionally or alternatively include otherelements and/or configurations of elements for generation of stableemulsions without requiring RI matching to have suitable clarity fordownstream analyses.

3. Method

As shown in FIGS. 6A and 6C, an embodiment of a method 300 forgeneration of an emulsion includes: generating a set of droplets from asample fluid 310; and generating the emulsion upon processing the set ofdroplets with a set of fluid layers 320 (e.g., to provide a thin filmabout each droplet, thereby forming at least a double emulsion), whereinthe set of fluid layers is configured with a density gradient. Invariations wherein processing the set of droplets includes dispersingthe set of droplets through the set of fluid layers, each fluid layer ofthe set of fluid layers is immiscible with adjacent fluid layers of theset of fluid layers, and at least one of the set of fluid layers can beconfigured to provide a thin film about individual droplets derived fromthe sample fluid, thereby producing clarity of the emulsion withoutrefractive index matching of components of the emulsion. In variations,the method 300 can additionally or alternatively include other suitablesteps associated with downstream applications. The method 300 functionsto provide benefits described in relation to Sections 1 and 2 above. Themethod 300 can be implemented using embodiments, variations, andexamples of the components of the system 100 described above or canalternatively be implemented using other suitable system components.

As shown in FIG. 6A, the method 300 includes generating a set ofdroplets of a sample fluid 310, where embodiments, variations, andexamples of system components for generating droplets from a samplefluid are described above. The set of droplets can be generated from anaqueous solution or a non-aqueous solution, where compositions ofaqueous and non-aqueous sample fluids are described above. Additionallyor alternatively, in variations, generating the set of droplets 310 caninclude implementing one or more of: microfluidic elements (e.g.,channels), needle instruments (e.g., stainless steel needle instruments,needles composed of another suitable/non-reactive material, etc.,capillary instruments (e.g., pulled glass capillary pipettes, capillaryplates, other capillary instruments, etc.), meshes, filter membranes,nebulizers, atomizers, electrodynamic droplet formation devices, and/orother suitable methods, where the droplets are then used to form theemulsion(s) described.

Alternatively, as shown in FIG. 6C, generating the set of droplets 310′can be performed according to another suitable method. Similarly, invariations, generating the set of droplets 310′ can include implementingone or more of: microfluidic elements (e.g., channels), needleinstruments (e.g., stainless steel needle instruments, needles composedof another suitable/non-reactive material, etc., capillary instruments(e.g., pulled glass capillary pipettes, capillary plates, othercapillary instruments, etc.), filter membranes, meshes, nebulizers,atomizers, electrodynamic droplet formation devices, and/or othersuitable methods, where the droplets are then used to form theemulsion(s) described. In variations, the generated droplets (e.g., as adispersed phase), can have a density greater than that of the continuousphase (i.e., first continuous phase described in more detail below) intowhich they enter. However, in other variations, the generated dropletscan have another suitable density.

3.1 Emulsion Generation—First Variation

As shown in FIG. 6A, the method 300 also includes generating an emulsionupon processing the set of droplets with a set of fluid layers 320. Inone variation, generating the emulsion can include dispersing the set ofdroplets through a set of fluid layers 320, where the set of fluidlayers is configured with a density gradient and wherein each fluidlayer of the set of fluid layers is immiscible with adjacent fluidlayers of the set of fluid layers. Step 320 functions to generateemulsions having suitable clarity without requiring RI matching betweenfluid components of the emulsion. In particular, Step 320 functions togenerate at least one stable thin film layer about each of the set ofdroplets generated from the sample fluid, where clarity of the resultantemulsions is due to the thickness of the thin film layers surroundingdroplets being less than the wavelength(s) of light used to observe theemulsions. In relation to step 320, the equilibrium position(s) of theset of droplets of the disperse phase at one or more of the set of fluidlayers also allows interrogation of the droplets (e.g., by using imagingapparatus) for downstream applications and assessments, and allowsseparation of the emulsion from layers (e.g., layers containingsurfactant micelles) that prevent clear observation of the emulsion atits equilibrium position.

In more detail, dispersing in step 320 can include applying a force(e.g., centrifugation, pressure, etc.) to the sample fluid in order todisperse the sample fluid through a substrate including a distributionof holes, followed by dispersion of generated droplets through one ormore of the set of fluid layers (i.e., layers of continuous fluidshaving a distribution of densities). The dispersed sample fluid isimmiscible with the first continuous fluid that it encounters but can bemiscible with the second continuous fluid it encounters. In variations,the dispersed fluid can have a density higher than the bottom-most fluidlayer, such that the generated droplets sink to the bottom of thecollecting container. The refractive indices of the various phases donot have to match, and the resultant emulsion is clear or otherwise hassuitable clarity for various applications described. Claritycharacteristics are achieved due to the continuous fluid(s) surroundingeach droplet is being thinner than the wavelength of light being used toilluminate the emulsion.

Embodiments, variations, and examples of configurations and compositionsof the set of fluid layers, as well as resultant emulsions, aredescribed in Section 2.3 above.

In a specific example of Step 320, as shown in FIG. 6B (top), W/O/Wemulsions were generated from sample fluids composed of water with anon-ionic surfactant and between 0% and 70% Optiprep™ (a densitygradient medium, however other density adjusting media can be used),where the sample fluids had various RIs between 1.33 and 1.40 (densities˜1 to >1 g/ml). The sample fluids were used to generate droplets usingan example of the system 100 described above, and the droplets weretransmitted through a set of fluid layers including an oil layercomposed of a silicone blend mixed with a silicone-based emulsifier(having a RI of 1.39, density <1 g/ml), prior to arrival at a continuousaqueous fluid layer of water mixed with an surfactant (having a RI of1.33, density˜1 g/ml). As shown in FIG. 7 (top), the emulsions hadsuitable clarity regardless of the final RI of any solution used togenerate the emulsions, but a distinct droplet layer did not form whenthe density of the sample fluid was equal to the density of the aqueousfluid layer used for the continuous phase. In comparison, FIG. 7(bottom) depicts an example where water-in-oil emulsions were generated,and where RI matching was required to have suitable clarity of emulsions(e.g., 59% Optiprep™ in water to match the RI of the oil-basedcontinuous phase).

3.2 Emulsion Generation—Second Variation

As shown in FIG. 6C, generating an emulsion upon processing the set ofdroplets with a set of fluid layers 320′ can include processing dropletsgenerated according to step 310′ with at least a second continuousphase, in order to generate a clear emulsion. In variations, the resultof step 310′ can be an emulsion of water-in-oil droplets (e.g., with anaqueous phase dispersed in a non-aqueous continuous phase as a firstcontinuous phase) or oil-in-water droplets (e.g., with a non-aqueousphase dispersed in an aqueous continuous phase as a first continuousphase), such that step 310′ produces an early stage emulsion havingclarity below a threshold level of clarity. In these variations, thedispersed phase has a density higher than that of the first continuousphase, and the result of step 310′ is an emulsion having low clarity dueto light scattering from the refractive index mismatches between thefluids (e.g., a cloudy emulsion).

In embodiments, clarity can be defined in units associated with clarityor turbidity (e.g., NTU, FNU), such that the threshold level of claritycan be measured for the emulsion(s) generated according to the methodsdescribed. In one variation, clarity can be characterized in relation totransmissivity as detectable by a transmission detector and/or inrelation to a suitable distance or depth (e.g., depth or distance into acollecting container for the emulsion; through a depth of a container ofthe emulsion, along an axis in which measurement of clarity isperformed, etc.), where, in variations, the threshold level of clarityof the emulsion is associated with a transmissivity greater than 70%transmissivity, greater than 80% transmissivity, greater than 90%transmissivity, greater than 95% transmissivity, greater than 99%transmissivity, etc. As such, in accordance with methods described, uponmeasuring clarity of the emulsion (e.g., later stage emulsion) using atransmission detector the emulsion is characterized by a clarityassociated with greater than 70% transmissivity, greater than 80%transmissivity, greater than 90% transmissivity, greater than 95%transmissivity, greater than 99% transmissivity, etc., which is abovethe threshold level of clarity.

Additionally or alternatively, in another variation, clarity can becharacterized in relation to clarity/turbidity as detectable by aturbidity measurement system (e.g., nephelometer, turbidimeter, etc.)and/or in relation to a suitable distance or depth (e.g., depth ordistance into a collecting container for the emulsion; through a depthof a container of the emulsion, along an axis in which measurement ofclarity is performed, etc.), where, in variations, the emulsion has aturbidity less than 10 turbidity units (e.g., nephelometric turbidityunits NTU)), less than 5 turbidity units, less than 1 turbidity unit,etc. As such, in accordance with methods described, upon measuringclarity of the emulsion using a turbidity measurement system theemulsion (e.g., later stage emulsion) is characterized by a clarityassociated with less than 10 NTU, less than 5 NTU, less than 1 NTU,etc., which is above the threshold level of clarity. Additionally oralternatively, turbidity can be characterized in terms of Formazinturbidity units (FTU), Helms units, parts per million, concentrationunits, optical density, and/or any other suitable units associated withany other suitable detection system.

After initial droplet formation according to step 310′, step 320′ caninclude processing the early stage emulsion with a second continuousphase that is immiscible with the first continuous phase and denser thanthe first continuous phase, in order to produce a later stage emulsionhaving clarity above a threshold level of clarity. Processing with thesecond continuous phase can include combining the second continuousphase with the early stage emulsion in a collecting container 322′, andapplying a force to the collecting container with the early stageemulsion and the second continuous phase to generate the later stageemulsion 323′.

In variations, the applied force can be a force applied bycentrifugation (e.g., at or above 10,000 G, below 10,000 G, at anothersuitable angular velocity, etc.), for a duration of time (e.g., lessthan or equal to one minute, greater than one minute, etc.). In aspecific example, applying the force in step 323′ can includecentrifuging the collecting container with the early stage emulsion andthe second continuous phase at 16,000 G for one minute; however,variations of the specific example can be implemented. Additionally oralternatively, the applied force can be a force applied bypressurization, vibration, rocking, or any other suitable method topromote movement of the second continuous phase for interaction withcontents (e.g., the dispersed phase, the first continuous phase) of thecollecting container and toward an equilibrium density gradient.

In variations, the second continuous phase is denser than the firstcontinuous phase, such that the applied force moves the secondcontinuous phase deeper into the collecting container and displaces anexcess volume of the first continuous phase in order to reduce physicaldistance between droplets in the dispersed droplet phase of the earlystage emulsion, thereby generating a later stage emulsion having clarityabove the threshold level of clarity.

In one example shown in FIG. 6D, the second continuous phase can bedenser than the first continuous phase but less dense than the disperseddroplet phase of the early stage emulsion, such that the excess volumeof the first continuous phase is displaced away from the disperseddroplet phase at the base of the collecting container in generating thelater stage emulsion having clarity above the threshold level ofclarity. In this example, the first continuous phase is a silicone oilblend with surfactants, the second continuous phase is intermediate indensity and composed of water (or a saline buffer) with a surfactant,and the dispersed droplet phase is a PCR mix with a density medium.

Alternatively, in another example shown in FIG. 6E, the secondcontinuous phase can be denser than the first continuous phase anddenser than the dispersed droplet phase of the early stage emulsion,such that the dispersed droplet phase settles away from the base of thecollecting container (e.g., between the first continuous phase and thesecond continuous phase, near an air interface, etc.) in generating thelater stage emulsion having clarity above the threshold level ofclarity.

Variations of the method 300′ can include processing intermediate stageemulsions with additional continuous phases (e.g., a third continuousphase, a fourth continuous phase, etc.) that are immiscible with eachother and/or other continuous phases being used, and having suitabledensity characteristics, in order to produce resultant emulsions havingeven greater clarity or other desired properties for variousapplications of use.

3.3 Emulsion Generation—Droplet Digital PCR

In variations, the method 300 can additionally or alternatively includeother suitable steps associated with downstream applications. Suchapplications can include performing one or more steps associated withPCR (e.g., thermocycling, incubating, mixing, imaging, materialretrieval, etc.), in order to analyze reactions or products generatedwithin each droplet of the disperse phase of the emulsion. For instance,as shown in FIG. 7A, the method 300 can include performing dropletdigital PCR with clear emulsions produced according to steps 310 and 320above. In particular, FIG. 7A depicts two cross-sectional images towardsthe center of a collecting container containing droplets that fluoresceafter performing droplet digital PCR, where the cross-sectional imagesobtained by light sheet imaging of the tube, where light sheet imagingcan be performed as described in PCT Application PCT/CN2019/093241 filed27 Jun. 2019, which is herein incorporated in its entirety by thisreference. In this example, the droplets were formed by generatingdroplets from 50 microliter of a PCR master mix containing polymerase,dNTPs, template DNA molecules, primers, dual-labeled hydrolysis probes.The droplets were generated by transmitting the mixture described abovethrough a 1 mm thick glass substrate having 37 microchannels, bycentrifugation at 16000 g. The formed droplets, as they exited the glasssubstrate, went into a 0.2 ml collecting container tube containing 50microliter of water with surfactant (as a continuous phase), above whichwas a layer of 50 microliter of silicone oil blend with siliconeemulsifier. Approximately 3.5 million drops were formed by this exampleprocess. In FIG. 7A, each fluorescent drop, measured to be ˜30 micron indiameter, represents amplification from one target molecule, and theclarity of the emulsion generated allowed capture of clear images alongeach the cross-sections of the tube. The number of fluorescent drops wasquantified to be ˜100 k.

3.3.1 Positive Droplet PCR

In particular, in comparison to conventional digital PCR platforms, inwhich the number of partitions are limited to the 20,000 or less andrequire counting both positive and non-positive compartments andapplying Poisson statistics to estimate counts, the invention(s)described herein do not require portioning error correctionmethodologies for counting applications (e.g., associated withdistributions of material), due to the high number of droplets generatedand operation at low occupancy (see FIG. 7B). As such, the invention(s)provide improved precision at high DNA counts with lower countinguncertainty. Such invention(s) can thus provide a droplet-based platformfor high dynamic range digital PCR. Applications of method(s) describedcan thus include droplet-based digital PCR without requiring counting ofnegative droplets (e.g., droplets not containing target material), andthereby without implementing correction factors (e.g., Poissoncorrection factors) for partitioning error, given that performance ofdigital PCR herein can be performed in a low occupancy (e.g., ˜5% orless total droplets are occupied by target material) regime. Inparticular, in embodiments, the method(s) can produce a high number ofdroplets (e.g., from 1 million to 100 million droplets) per unit volume(e.g., 10 microliter to 100 microliter), each droplet having acharacteristic diameter (e.g., 10 micron-100 micron). In one example,the method(s) can produce a high number of droplets (e.g., 3.5 milliondroplets having a characteristic droplet diameter of 30 micron within a50 microliter volume). In another example, the invention(s) can producea high number of droplets (e.g., 28 million droplets having acharacteristic droplet diameter of 15 um within a 50 ul volume).However, variations can produce other numbers of droplets (e.g., greaterthan 500,000 droplets/partitions) having other suitable characteristicdiameters within other suitable collection volume sizes.

In particular, with respect to droplet digital PCR, methods can includesteps for detecting and quantifying only droplets emitting a positivesignal (e.g., with respect to target material sequences), withoutdetecting and/or quantifying droplets having a negative signal. As such,methods described can be used for positive digital PCR, withoutrequiring detection or quantification of droplets having a non-positivesignal.

As shown in FIG. 7C, such methods can thus include: a method 400 ofcounting nucleic acids in a sample (e.g., a sample of more than 5,000nucleic acids, a sample of more than 10,000 nucleic acids, a sample ofmore than 20,000 nucleic acids, etc.) wherein said nucleic acidsdistributed across a set of partitions 410 (e.g., within droplets of anemulsion, partitioned in another manner), and wherein only a subset ofthe set of partitions emitting signals associated with said nucleicacids (i.e., droplets providing positive readout) are counted (anddroplets not providing positive readout are not counted) 450.

Relatedly, as shown in FIGS. 7D and 7E, a method 400′ for performingdroplet digital PCR can thus include: generating a set of droplets froma sample fluid 410′; generating an emulsion having clarity above athreshold level of clarity upon processing the set of droplets with oneor more fluid layers configured with a density gradient, wherein eachfluid layer of the one or more fluid layers is immiscible with adjacentfluid layers of the one or more fluid layers, thereby producing clarityof the emulsion without refractive index matching of the sample fluidand the one or more fluid layers 420′; inducing one or more reactionsupon processing the emulsion with a set of operations 430′; detectingsignals emitted from a subset of droplets containing target materialfrom the sample fluid 440′; and performing a quantification operationwith the subset of droplets 450′.

Generating the set of droplets and generating the emulsion in steps 410and 420′, respectively, can be performed according to methods (e.g.,method 300, method 300′) described above and/or according to othersuitable methods.

Furthermore, as described above, in embodiments, the sample fluid caninclude target material (e.g., nucleic acids), and in specific examples,the droplets can be generated from a sample fluid with a PCR master mixcontaining polymerase, dNTPs, template DNA molecules, primers, probesand/or a solute (e.g., sugar or salt component). As such, generation ofdroplets according to the method 400′ can produce an emulsion havingsuitable clarity, where some, but not all droplets generated contain thetarget material. Inducing one or more reactions upon processing theemulsion in step 430′ can thus include performing one or more stepsassociated with amplification and detection of nucleic acids (e.g.,thermocycling, incubating, mixing, etc.) within the collectingcontainer.

Post-reaction, the method 400 can include detecting signals emitted froma subset of droplets containing target material from the sample fluid440′, where positive signals are associated with presence of targetmaterial captured within individual droplets of the subset of droplets.The detected signals are preferably optically-detectable (e.g., using anoptical detection subsystem, using an imaging subsystem, etc.), whereembodiments, variations, and examples of an optical detection subsystemare described in in PCT Application PCT/CN2019/093241 filed 27 Jun.2019, incorporated by reference above; however, in variations, thesignals can be detectable in another suitable manner by other suitableapparatus.

As shown in FIG. 7D, the method 400′ includes performing aquantification operation with the subset of droplets in step 450′. Inparticular, performing the quantification operation can be implementedin a manner such that only the subset of droplets that positively emitsignal associated with target material are counted according to thequantification operation, without counting of droplets that do notpositively emit signal. As such, in contrast to other droplet-basedapproaches, the method 400′ can omit counting of droplets that do notpositively emit signal associated with the target material, as shown inFIG. 7 , thus providing an improved and more efficient process withrapid generation of results.

In specific applications, above-described methods for droplet digitalPCR can be used for or adapted for (e.g., in relation to analytes otherthan nucleic acids and reactions other than PCR) one or more of:enumeration of protein or peptide molecules (e.g., by proximity ligationassays, etc.); sequencing applications (e.g., single molecule sequencingapplications); monitoring or detection of products (e.g., proteins,chemicals) released from single cells (e.g., interleukin released fromimmune cells); monitoring cell survival and/or division for singlecells; monitoring or detection of enzymatic reactions involving singlecells; antibiotic resistance screening for single bacteria; enumerationof pathogens in a sample (e.g., in relation to infections, sepsis, inrelation to environmental and food samples, etc.); enumeration ofheterogeneous cell populations in a sample; enumeration of individualcells or viral particles (e.g., by encapsulating cells in droplets withspecies-specific antibodies coupled with enzymes that react withsubstrate components in the droplet to produce signals, etc.);monitoring of viral infections of a single host cell; liquid biopsiesand companion diagnostics; prenatal diagnosis of genetic disorders(e.g., aneuploidy, genetically inherited diseases) such as withcell-free nucleic acids, fetal cells, or samples containing mixtures offetal and maternal cells based upon generated counts and subsequentcharacterization of target nucleic acids; detection of cancer forms fromvarious biological samples (e.g., from cell-free nucleic acids, tissuebiopsies, biological fluids, faeces) based upon generated counts andsubsequent characterization of target nucleic acids; detection and/ormonitoring of minimal residual diseases; monitoring responses totherapies; detection or prediction of rejection events of transplantedorgans; other diagnostics associated with other health conditions; othercharacterizations of statuses of other organisms; and other suitableapplications.

3.4 Emulsion Generation—Additional Applications

Variations of the method 300 can additionally or alternatively includeother steps involving adjustments to the temperature(s) of emulsionsgenerated, where the emulsions are stable across temperature ranges inuse. In terms of stability, the components of the disperse phase and thecontinuous phase of the emulsion can be configured such that the film(s)about each droplet of the disperse phase are stable and do not break orresult in droplet coalescence across temperature ranges in use. Inparticular, temperature adjustments can be associated with thermocycling(e.g., for amplification processes), temperatures for cell cultureapplications, temperatures storage (e.g., at refrigerated temperatures,at ambient temperatures), and/or other temperatures used forapplications in other fields (e.g., pharmaceutical applications, foodproduction applications, etc.). In a specific example, the emulsiongenerated is stable for across material storage and thermocyclingapplications, for instance, between 4 C and 95 C (e.g., for 20-30minutes).

Additionally or alternatively, in some variations, the method 300 caninclude steps associated with performance of multi-step reactions oranalyses. For instance, in relation to generating emulsions, the method300 can include passing droplets of the dispersed phase through multiplelayers of a set of fluid layers, where each layer includes componentsassociated with a reaction (e.g., tagging with a moiety, generation of aproduct, etc.). In one such example, a components of a droplet can “pickup”, bind to, or otherwise react with content of each fluid layerencountered, to perform multi-step reactions.

In another variation, the method 300 can additionally or alternativelyinclude steps for receiving additional fluids into the collectingcontainer after an initial emulsion is generated. For instance, after aninitial emulsion is generated and/or a product is generated from areaction associated with components of the emulsion, the method 300 caninclude receiving one or more additional material components into thecollecting container (e.g., through an opening, through a valvedconnection into the collecting container, etc.) where the additionalmaterial components can be configured to react with or interact with theemulsion in some manner for a downstream application.

However, the method 300 can additionally or alternatively include othersteps for generating, processing, and/or using emulsions having suitableclarity for at least one stage of processing.

3.5 Method—Specific Example for Antimicrobial Susceptibility Testing

In specific example applications, clear emulsions generated by themethod 300 can be used for antimicrobial susceptibility testing, where asample fluid can include a fluorescent compound reactive with targetmaterial of the sample fluid. The method can then include performingfluorescent imaging of the emulsion generated from the sample fluid,within the collecting container, and processing readout signals fromindividual droplets of the emulsion within the collecting container. Inmore detail, in a specific example, a solution containing bacteria andan antibiotic for susceptibility testing can be combined with afluorescent growth indicator (e.g., resazurin) and/or a live-deadstaining component. The solution can then be processed to generatedroplets (e.g., by centrifugation through an example of the system 100described above) the droplets. In this example, the amount of bacteriaused is configured to be smaller than the number of droplets generated,such that each droplet contains one or zero bacterium units, withantibiotic components. In the example, antibiotic efficacy is assessedby imaging droplets, as droplets containing live/growing and deadbacterium units will fluoresce according to the live-dead stain. Imagingof droplets is thus enabled due to the transparency of the emulsion(e.g., having greater than 80% optical transmittance of light, havinganother suitable level of light transmittance, etc.), which enablesreading of fluorescence of individual droplets throughout the tube(e.g., using confocal imaging, using light sheet imaging). Furthermore,due to the small volume of each droplet of the disperse phase of theemulsion, signals can be observed after only a few cell replicationcycles. This reduces turn-around time for results from a few days oftypical plate-based culture to a few hours or less. The digital read-outalso enables one to monitor heterogeneous response to antibiotic of themicrobial community in the sample.

Alternative to fluorescent monitoring of growth associated dyes in theexample described above, an increase in number of cells within a dropletand/or generation of reaction products can cause the droplet to havedifferent light scattering properties. As such, droplet turbidity can bemeasured quantitatively by a turbidimeter or spectrophotometer, or evenby visual observation.

The method 300 can, however, be applied to other specific applicationsin relation to processing and/or analyzing sample emulsions.

4. Conclusions

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or, if applicable, portion ofcode, which comprises one or more executable instructions forimplementing the specified logical function(s). It should also be notedthat, in some alternative implementations, the functions noted in theblock can occur out of the order noted in the FIGURES. For example, twoblocks shown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the block diagrams and/or flowchart illustration, andcombinations of blocks in the block diagrams and/or flowchartillustration, can be implemented by special purpose hardware-basedsystems that perform the specified functions or acts, or combinations ofspecial purpose hardware and computer instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

What is claimed is:
 1. A method comprising: performing a digitalanalysis of a sample distributed across a set of partitions, whereinperforming the digital analysis comprises imaging a set ofcross-sections of a closed container containing the set of partitions,wherein the set of partitions has a number greater than 1 millionpartitions, and wherein the digital analysis is performed within threeminutes.
 2. The method of claim 1, wherein the digital analysis isperformed within two minutes.
 3. The method of claim 1, wherein imagingthe set of cross-sections of the closed container comprises performingreadout of signals from contents of partitions represented in each ofthe set of cross-sections.
 4. The method of claim 3, wherein the signalscomprise fluorescent signals from labelled target analytes of thesample.
 5. The method of claim 1, wherein the set of partitionscomprises a set of droplets of an emulsion generated from the sample,wherein clarity of the emulsion is produced without refractive indexmatching of the sample and other materials of the emulsion.
 6. Themethod of claim 5, wherein each of the set of droplets has acharacteristic diameter from 10 microns to 100 microns.
 7. The method ofclaim 5, wherein the set of droplets is characterized by less than 5%occupancy by target analytes of the sample.
 8. The method of claim 1,wherein the closed container has a volume from 10 to 100 microliters. 9.The method of claim 1, wherein performing the digital analysis comprisesanalyzing up to 1.4 million nucleic acid molecules of the sample withoutapplication of Poisson statistics to correct for partitioning error. 10.The method of claim 1, wherein the sample comprises a nucleic acidmixture combined with a solution for polymerase chain reaction (PCR) anda density medium.
 11. The method of claim 10, wherein the nucleic acidmixture comprises cell-free nucleic acids.
 12. The method of claim 1,wherein the sample comprises material derived from fetal cells andmaternal cells, and wherein performing the digital analysis furthercomprises performing prenatal detection of aneuploidy.
 13. The method ofclaim 1, wherein the sample comprises a protein solution.
 14. The methodof claim 1, wherein imaging the closed container comprises performing a3D scanning technique.
 15. The method of claim 1, wherein imaging theclosed container comprises performing a planar imaging technique.
 16. Amethod comprising: performing a digital analysis of a set of dropletsgenerated from a sample and retained at equilibrium positions within acontinuous fluid layer within a closed container, wherein performing thedigital analysis comprises scanning one or more cross-sections of theclosed container, wherein the set of droplets has a number greater than1 million droplets, and wherein the digital analysis is performed withinthree minutes.
 17. The method of claim 16, wherein each of the set ofdroplets has a characteristic diameter greater than 1 micron.
 18. Themethod of claim 16, wherein scanning one or more cross-sectionscomprises performing at least one of a 3D scanning technique and aplanar imaging technique.
 19. The method of claim 16, wherein: thesample comprises a first aqueous solution comprising target nucleicacids, a mixture for polymerase chain reaction (PCR), and a densitymedium, the continuous fluid layer comprises a second aqueous solution,and the set of droplets generated from the sample are individuallyisolated and separated from the second aqueous solution by a non-aqueoussolution.
 20. The method of claim 19, wherein performing the digitalanalysis further comprises performing a temperature adjustment operationon the set of droplets at the equilibrium positions, wherein the set ofdroplets is thermally stable during the temperature adjustmentoperation.
 21. A method comprising: performing a digital analysis of aset of droplets generated from a sample and retained at equilibriumpositions within a continuous fluid layer within a closed container,wherein performing the digital analysis comprises scanning a set ofcross-sections of the closed container, wherein the set of dropletscomprises 3.5 million droplets, wherein the digital analysis isperformed within three minutes, and wherein performing the digitalanalysis comprises performing prenatal detection of a genetic disorder.